An in‐house 45‐plex array for the detection of antimicrobial resistance genes in Gram‐positive bacteria

Abstract Identifying antimicrobial resistance (AMR) genes and determining their occurrence in Gram‐positive bacteria provide useful data to understand how resistance can be acquired and maintained in these bacteria. We describe an in‐house bead array targeting AMR genes of Gram‐positive bacteria and allowing their rapid detection all at once at a reduced cost. A total of 41 AMR probes were designed to target genes frequently associated with resistance to tetracycline, macrolides, lincosamides, streptogramins, pleuromutilins, phenicols, glycopeptides, aminoglycosides, diaminopyrimidines, oxazolidinones and particularly shared among Enterococcus and Staphylococcus spp. A collection of 124 enterococci and 62 staphylococci isolated from healthy livestock animals through the official Belgian AMR monitoring (2018–2020) was studied with this array from which a subsample was further investigated by whole‐genome sequencing. The array detected AMR genes associated with phenotypic resistance for 93.0% and 89.2% of the individual resistant phenotypes in enterococci and staphylococci, respectively. Although linezolid is not used in veterinary medicine, linezolid‐resistant isolates were detected. These were characterized by the presence of optrA and poxtA, providing cross‐resistance to other antibiotics. Rarer, vancomycin resistance was conferred by the vanA or by the vanL cluster. Numerous resistance genes circulating among Enterococcus and Staphylococcus spp. were detected by this array allowing rapid screening of a large strain collection at an affordable cost. Our data stress the importance of interpreting AMR with caution and the complementarity of both phenotyping and genotyping methods. This array is now available to assess other One‐Health AMR reservoirs.


| INTRODUCTION
Antimicrobial resistance (AMR) has become a major concern threatening public health (Nowakiewicz et al., 2019). For many decades, antibiotics are widely used in animal and human areas, leading to the worldwide resistance phenomenon. Indeed, bacteria have always been able to adapt by developing or acquiring mechanisms of resistance (Duval et al., 2019). In response to selective pressure and to survive antibiotic exposure, resistance occurs generally through the acquisition of genes located on mobile elements (Argudin et al., 2017;Strauss et al., 2015).
Despite awareness associated with restrictive measures in animal production, the effects of the intensive use of drugs seem to persist lengthily, impacting numerous environments and ecological niches (Argudin et al., 2017;Nowakiewicz et al., 2019).
Commensal bacteria, such as Enterococcus spp. are natural inhabitants of the gastrointestinal tract of healthy animals and humans. Enterococci can also be considered opportunistic pathogens when found related to human infections. They can persist in the environment and survive in different ecological niches such as soil, water, food items, and sewage (Nowakiewicz, 2019;Osman et al., 2019;Raza et al., 2018;Torres et al., 2018). Enterococci have emerged as one of the four most prevalent nosocomial human pathogens worldwide, especially due to their virulence, ability to form a biofilm, and intrinsic resistance (Leite-Martins et al., 2015;Raza et al., 2018). Notably, Enterococcus spp. are intrinsically resistant to a number of antimicrobials including trimethoprim-sulfamethoxazole, vancomycin (Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens), streptogramins (Enterococcus faecalis) and exhibit low-level resistance to ß-lactams and aminoglycosides (Argudin et al., 2017;Torres et al., 2018;Zaheer et al., 2020). Since the 1980s, AMR enterococci have been the leading cause of hospitalacquired bloodstream and urinary tract infections, mainly through biofilm formation on catheters and implanted medical devices (Argudin et al., 2017;De Jong et al., 2019;Mercuro et al., 2018;Raza et al., 2018;Torres et al., 2018;Zaheer et al., 2020). Particularly, the majority of enterococcal infections are caused by E. faecalis and Enterococcus faecium, the most common species encountered in the human gut (Argudin et al., 2017;Mercuro et al., 2018;Raza et al., 2018;Torres et al., 2018). E. faecium has become a prominent cause of nosocomial infections often characterized by high-level resistance to multiple antibiotics (De Jong et al., 2019). Due to the high plasticity of enterococcal genomes, transfer and acquisition of AMR determinants in enterococci and other Gram-positive bacteria are then facilitated (Leite-Martins et al., 2015;Nowakiewicz et al., 2019).  Wendlandt et al., 2013). Despite preventing colonization by pathogenic bacteria (Alharbi, 2019), staphylococci are opportunistic pathogens often responsible for chronic and severe nosocomial infections (Bortolaia et al., 2016;Dastgheyb & Otto, 2015).
Specifically, Staphylococcus aureus is one of the most pathogenic bacteria associated with human and animal diseases causing persisting skin and soft tissue infections (SSTIs), infectious endocarditis, septic arthritis, and osteomyelitis (Alharbi, 2019;Craft et al., 2019;Dastgheyb & Otto, 2015). The colonization of skin or mucosal surfaces by methicillin-resistant S. aureus (MRSA) and its dissemination in healthcare settings represents a global health issue (Holmes et al., 2015;Watkins et al., 2019) especially due to its capacity to acquire new AMR and to spread rapidly (Alharbi, 2019;Holmes et al., 2015). Indeed, methicillin resistance was first observed among clinical isolates before its rapid spread to the community (Turner et al., 2019). Staphylococcus spp. may exchange resistance determinants to numerous other bacteria in the same animal or human host, or between hosts by direct contact or through excretions such as sneezing, coughing, or licking . Even if MRSAassociated infection rates have declined, these human infections are still problematic since they are characterized by broadening AMR and high rates of hospitalization and mortality (Purrello et al., 2016).
Resistance to last-resort drugs was already observed as well (Azhar et al., 2017;Doern et al., 2016;Holmes et al., 2015;Purrello et al., 2016) and among various bacteria, mainly reported in clinical settings (Argudin et al., 2017;Zaheer et al., 2020). Specifically, due to the alarming worldwide emergence (Osman et al., 2019;Raza et al., 2018), vancomycin-resistant enterococci (VRE) was ranked as a pathogen of high priority by the World Health Organization (WHO) (Wist et al., 2020). Due to its ability to confer high levels of vancomycin-resistance, vanA is the most reported gene among clinical VRE (Azhar et al., 2017;Turner et al., 2019;Watkins et al., 2019) and widely spread to other co-infecting bacteria such as S. aureus. Despite restricted use, resistance to linezolid has been reported in various species, strains, and settings (Sadowy, 2018).
In summary, AMR is not restricted to a particular species or host. It became essential to consider all bacteria as a potential pool of resistance determinants possibly transferable to other pathogenic or commensal bacteria both in animals and humans (Argudin et al., 2017;De Jong et al., 2019;Osman et al., 2019;Perreten et al., 2005;Torres et al., 2018;Zaheer et al., 2020). Therefore, it is important to identify AMR determinants spreading in humans and animals (Perreten et al., 2005;Strauss et al., 2015) and to consider them as a One Health AMR pool. The complexity of AMR and particularly the cross-resistance phenomenon, that is resistance to multiple distinct antimicrobial classes conferred by a single molecular mechanism, requires monitoring all putative main sources of AMR at the genetic level.
We describe an in-house developed array targeting major AMR genes of Gram-positive bacteria and allowing their rapid and efficient detection all at once at reduced costs. In this study, we aimed to target AMR genes commonly found and particularly shared among Enterococcus and Staphylococcus spp. The diversity of probes per antimicrobial class reflects phenotypes observed during several years of field monitoring and pointed out the most critical (e.g., glycopeptides) or emergent ones (e.g., linezolid) to investigate. The presence of these AMR genes was studied in a collection of 124 enterococci and 62 staphylococci isolated from healthy livestock animals through the official Belgian AMR monitoring during the period 2018-2020, to decipher the genetic nature of this pool of AMR and thereby provide useful data to understand how resistance can be acquired and maintained in these bacteria.

| PLPs probes design
Probes were designed as described previously (Boland et al., 2018;Timmermans et al., 2022b;Wattiau et al., 2011). The sequences of the probes developed in this study and used in the LCR assay and the corresponding AMR genes targeted by the array are listed in Enterococcus and Staphylococcus spp. were aligned with Bionumerics 6.6 (bioMérieux SA) and target-specific sequences were selected within the most conserved regions. In addition to these specific sequences corresponding to the two extremities of the probes, the socalled 5'-arms and 3'-arms, probes encompassed: sequences of the universal primers "reverse" (cUR) and "forward" (UF; see Table 2) as well as an anti-TAG sequence matching a given TAG sequence of the MagPlex-TAG™ microspheres (Luminex). Probes are schematically represented in linear form as follows: 5'arm -cUR -AA -UFanti-TAG -3'arm where "AA" is a di-deoxyadenosine linker.
Right after hybridization, three washes were performed by pelleting the beads on a magnetic bead separation system (V&P

| Data analysis
The fluorescence signal of the Gram-positive control probe "Gram+" was used as an internal standard to normalize MFI signals observed for each sample according to the formula: (MFI probe/MFI Gram+) × 100. The results expressed in normalized MFI (nMFI) were evaluated against cut-offs. Cut-offs were determined after plotting experimental nMFI values obtained by testing isolates and control strains and included a twofold ratio between positive and negative nMFI. The probes of the array were validated with reference strains used as positive controls, except for the vgaD probe for which no reference strain was available (see Table A1). The control probes soda_fs and soda_fm are based on the housekeeping sodA gene of E.

| RESULTS
In this study, a bead array based on AMR genes was developed and validated on reference strains (see Table A1) to characterize the genetic nature of resistance expressed by Gram-positive bacteria.  (Wattiau et al., 2011;Boland et al., 2018); normal characters indicate nucleotides added to reach a final set of probes with evenly distributed sizes ranging from 99 to 125 nucleotides. For the design of each probe, the most conserved region of the targeted gene, obtained from alignments in Bionumerics© of sequences available in GenBank, was chosen as the target sequence. Three control probes (indicated with the letter "C") were designed for the identification of each bacterial species, Enterococcus faecalis, Enterococcus faecium, and Staphylococcus aureus. a Nucleotide sequence including a wobble.    and vgbB, are also involved in streptogramins A or B resistance (see Table 1) (Cho et al., 2020;De Graef et al., 2007;Pechère, 2001;Petinaki & Papagiannitsis, 2019;Roberts, 2008) were screened with this array, but not detected. One of the vat genes, namely vatD, was Noteworthy, the Q/D resistance mechanisms are still not perfectly understood. Indeed, few studies reported that resistance to streptogramin A is sufficient to confer resistance to Q/D (Hancock, 2005;Yan et al., 2021) while others support that a combination of both resistance to streptogramin A (vat or vga family) and streptogramin B (erm family or vgbB) is required to ensure Q/D resistance (Miller et al., 2014;Zarrouk 2000). Both lsaA and lsaE are the only genes reported to confer resistance to both Q/D components (Alcock et al., 2020;Singh et al., 2002;Wendlandt et al., 2013). In this study, the presence of lsaA, lsaE, or a combination of both streptogramin A and B resistance genes has been considered concordant with a Q/D resistance phenotype.

| Macrolide resistance
Target modification by erm genes (coding for 23S rRNA methylases) is known to confer macrolide, lincosamide, and streptogramin B lsaA 57 (100.0%) 0 (0.0%) 0 (0.0%) vanC 2-3 0 (0.0%) 0 (0.0%) 0 (0.0%) Note: For each antimicrobial, the numbers in the first line correspond to the number of isolates that were phenotypically resistant to this antimicrobial and assessed with the array. In front of each gene, the numbers in each column indicate the number of isolates in which this gene was detected with the array and the corresponding percentage of isolates carrying this gene among the number of isolates indicated in the top line for each antimicrobial. A dash indicates that the presence of the gene in the corresponding bacterial species was not assessed because the phenotypic resistance profile was not available.

| Pleuromutilin resistance
Tiamulin resistance reported in 35 S. aureus is known to be conferred by lsa, cfr, or vga genes Schwarz et al., 2018;Van Duijkeren et al., 2014). In this study, 82.9% of tiamulin-resistant isolates harbored lsaE (n = 28) alone or in combination with cfr (n = 1), while lsaA and vga genes were not detected. Six tiamulin-resistant isolates did not harbor any relevant gene. AST for tiamulin resistance was not assessed in enterococci.

| Tetracycline resistance
Although 59 different tetracycline resistance genes have been described (Marosevic et al., 2017), only the most frequent, tetM and tetO mediating resistance through ribosomal protection and tetK and tetL mediating resistance through efflux, were included in this study (Cho et al., 2020;Perreten et al., 2005). In the investigated enterococci isolates, tetracycline resistance was mainly characterized by the presence of tetM or a combination of tetM/tetL genes. Indeed, tetM was identified in 90.5% and 100% of the tetracycline-resistant E. faecalis (n = 48/53) and E. faecium (n = 65/65), respectively.
All tetracycline-resistant isolates from this study harbored at least one of the four targeted tet genes. More specifically, tetL was always found in combination with other tet genes, with tetM (n = 91) or tetM/ tetO (n = 2) in enterococci, and tetM (n = 12) or tetM/tetK (n = 11) in staphylococci. In addition, tetL and tetM were found on the same contig in 19 of 25 (76.0%) sequenced enterococci, adjacent in 18 of them. In the latter, tetL and tetM were separated by~3 kb but no insertion sequence (IS) was found between the two genes.  fexA was observed in this study in 81.3% (n = 26/32) of the enterococcal isolates, as reported elsewhere (Brenciani et al., 2018;Ruiz-Ripa et al., 2020;Sadowy, 2018;Timmermans et al., 2022a). In addition, fexB was not included in the array but was detected in 10 sequenced enterococcal isolates, including five chloramphenicolsusceptible strains characterized by a MIC of 32 mg L −1 . fexB was detected together with poxtA (n = 3), optrA/poxtA (n = 5), or fexA/optrA/poxtA (n = 2). Genome analysis indicated that the optrA/ fexA combination was found on the same contig in 87.5% of isolates (n = 15/16). optrA and fexA were close to each other (~700 bp) in 14 of them and more distant in the remaining isolate (~5 kb). No IS was found between optrA and fexA in any of these isolates. The poxtA/ fexB combination was never associated with the same contig, as described elsewhere (Freitas et al., 2020;Ruiz-Ripa et al., 2020;Timmermans et al., 2022a). As expected, vanC 1 and vanC 2-3 , intrinsic to E. gallinarum and E. casseliflavus, respectively, were not detected. Vancomycin resistance being absent in staphylococci from the studied years, no

| Glycopeptide resistance
vancomycin-resistant isolates were tested with this array and in line with this, no van genes were detected with the array.

| Comparison of array-genotype with WGS-genotype: Short investigation
The AMR genetic profile of 31 isolates (16 E. faecalis, 14 E. faecium, and 1 MRSA) was investigated by whole-genome sequencing and compared to data resulting from the array. The selection of isolates for WGS was based on resistance to linezolid (n = 22), vancomycin (n = 2), or a minimum of 5 antimicrobials (n = 7). The genetic profiles gathered with the AMR array were all confirmed by WGS analyses (31/31) (see Table 5). These results ensured the reliability of the array method. Conversely, WGS investigation highlighted genes not covered by this array, that is vanL found in one vancomycinresistant isolate (VAR-660). 78.4%), as well as to tiamulin (n = 29/35; 88.6%) and clindamycin (n = 29/57; 50.9%). Such pleuromutilin-lincosamide-streptogramin A resistance mediated by lsaE as found in our study among Staphylococcus spp. and Enterococcus spp. isolated from animals was reported elsewhere in animals and also in humans Schwarz et al., 2018;Wendlandt et al., 2013).
All tetracycline-resistant isolates of this study harbored at least one of the tested genes, namely tetL, tetM, tetK, and/or tetO, with In this study, at least one linezolid-resistance gene was detected in each LZD-R strain: optrA, poxtA, or a combination of both genes in 63.6%, 9.1%, and 27.3% of LZD-R enterococcal isolates, respectively.
These genes were also found in LZD-susceptible isolates characterized by a MIC of 4 mg L -1 (n = 9/15), 2 mg L -1 (n = 5/60), or 1 mg L -1  presence of a resistance gene such as fexA (n = 4) and cat pC194 (n = 7) as described for LZD-susceptible isolates here above. cat pC194 , cat pC221 , and cat pC223 from the cat enzyme family as well as fex exporters are inducible (Schwarz et al., 2016), which could explain the variations observed among MICs. This highlights the limitations of AST based on phenotypic cut-offs for the screening of AMR genes, in particular for such inducible genes. In addition, future genetic investigation of chloramphenicol phenotypic resistance may partially rely on the detection of fexB, a member of the fex exporters family already reported in enterococci (Argudin et al., 2017;Schwarz et al., 2018) and found in 10 sequenced enterococci of this study.
Interestingly, the concomitant presence of optrA and fexA was observed in 23 of our isolates independently of phenotypic resistance profiles. Particularly, optrA was found close to fexA in 14 of the 16 isolates investigated by WGS. The absence of IS between the two genes in these isolates suggests both genes might be transferable together.
Vancomycin resistance, although rare, was characterized by the presence of the vanA cluster in one of the two isolates, the most common gene reported in other studies (Courvalin, 2006;Ekwanzala et al., 2020;Torres et al., 2018). In the remaining vancomycin-resistant isolate (VAR-660), sequencing highlighted the presence of the rare vanL, a gene not covered by this array. The rare vanL gene cluster, first described by Boyd et al. (2008), has been so far detected on the chromosome of a single E. faecalis isolate of human origin (Ekwanzala et al., 2020) displaying low-level vancomycin resistance (Ekwanzala et al., 2020;Boyd et al., 2008) as observed here (MIC = 8 mg L −1 ).
Our study reports the presence of vanL in an E. faecalis isolate from animal origin. The origin of the vanL gene cluster and the way an animal (i.e., a pig) acquired this strain remain elusive.
In a few cases of this study (10.8% of the individual resistant phenotypes in staphylococci and 7.0% in enterococci), a genetic marker could not be associated with the resistant phenotype. In enterococci, no relevant AMR genes were found with the array to explain erythromycin (n = 3), Q/D (n = 6), chloramphenicol (n = 10), gentamicin (n = 1) and vancomycin (n = 1) phenotypic resistances (out of a total of 444 assessed phenotypes). In staphylococci, a genetic explanation was missing for clindamycin (n = 1), erythromycin (n = 6), Q/D (n = 2), tiamulin (n = 6), chloramphenicol (n = 2), gentamicin (n = 2), streptomycin (n = 7), and trimethoprim (n = 7) resistant phenotypes (out of a total of 342 assessed phenotypes). In addition, the results of this study highlighted the complexity of Q/D resistance and a lack of a complete genetic explanation in 14/90 isolates since the identified genes (except for lsaA and lsaE) have been reported to confer resistance to either streptogramin A or streptogramin B but not both. Particularly, the presence of erm (streptogramin B) alone in Q/D-resistant isolates (10/53 E. faecium, 4/37 MRSA) does not explain the observed Q/D-resistant phenotype. Similar to lsa, eatA would be an interesting target as this gene confers the same profile of cross-resistance to lincosamides, streptogramins A, and pleuromutilins (Isnard et al., 2013). The WGS investigation revealed the presence of msrC in one E. faecium (VAR-681) exhibiting resistance to Q/D, this gene being frequently reported elsewhere in this species (Frye & Jackson, 2013;Hollenbeck & Rice, 2012). And, msrC has been described in Q/D-susceptible isolates, however first reported as being specifically found in resistant E. faecium. This suggests that it might be silent or involved in resistance to other antibiotics (Smoglica et al., 2022) such as macrolides (Portillo et al., 2000;Zaheer et al., 2020).
Therefore, msrA which shares significant sequence identity with msrC has been reported to confer macrolides-streptogramin B resistance in staphylococci and could be an interesting future target to investigate erythromycin and/or Q/D phenotypic resistances (Reynolds & Cove, 2005). In addition, ermF, ermY, mphB or more recently described mefD, msrF, and msrH have been found in staphylococci to confer macrolide and lincosamide resistances (Miklasinska-Majdanik, 2021;Schwarz et al., 2018;Woodford, 2005). Besides, ereA and ereB esterases have been reported to confer macrolide resistance in staphylococci isolated from animals as well (Miklasinska-Majdanik, 2021;Schwarz et al., 2018). In addition, chromosomal mutations in rplD or rplV coding for ribosomal proteins L4 and L22 leading macrolide and Q/D resistance, respectively, were also observed in Streptococcus pneumoniae and S. aureus and could be targeted in future studies (Farrell et al., 2004;Malbruny et al., 2002;Miklasinska-Majdanik, 2021). The sal gene family reported to confer lincosamide, streptogramin A and pleuromutilin resistance in staphylococci from humans or animals at various levels could be investigated in an attempt to explain the tiamulin-resistant phenotypes observed in 6/35 isolates (Mohamad et al., 2022;Schwarz et al., 2018).
Pleuromutilin resistance also derives from chromosomal mutations in 23S rRNA or rplC as described in staphylococci isolated from both humans and animals (Paukner & Riedl, 2017;Van Duijkeren et al., 2014). No gene of this array was detected in 10 isolates (1 E. faecalis and 9 MRSA) exhibiting resistance to aminoglycosides, namely to gentamicin (n = 2 MRSA and 1 E. faecalis) and to streptomycin (n = 7 MRSA). In enterococci, an intrinsic low-level of aminoglycoside resistance can be occasionally observed due to the presence of the species-specific chromosomal aac(6')-Ii gene found in almost all E. faecium (Adamecz et al., 2021). Among many aminoglycosides modifying enzymes (AMEs) described in the literature, a few of them such as str could be interesting to target in the future as already described in staphylococci (Ramirez & Tolmasky, 2010;Schwarz et al., 2018). In contrast, few cases of mismatch were observed with kanamycin-susceptible isolates (n = 7 MRSA) by BMD testing carrying one gene encoding aminoglycosides resistance (aadD in this study), as already reported in other studies (Adamecz et al., 2021;Feizabadi et al., 2006;Yean et al., 2007). This could be explained if this gene is nonfunctional and could be investigated in future studies. In addition, Ida et al. (2002) have shown that rearrangements caused by the KOWALEWICZ ET AL. | 15 of 21 integration of insertion elements into the staphylococcal chromosome or plasmids affected the expression of adjacent genes including aminoglycoside resistance genes. Finally, 7/55 (12.7%) trimethoprimresistant MRSA isolates were not found to be associated with the presence of a genetic marker in this study. The presence of other members of the dfr genes family (e.g., dfrF) or the presence of chromosomal mutations may explain the phenotypic resistance observed in these isolates (Woodford, 2005).

| CONCLUSIONS
In this study, a bead array was described aiming to detect broad AMR genetic profiles of Gram-positive bacteria from healthy animals in a single experiment. A number of resistance genes circulating among Enterococcus spp. and Staphylococcus spp. were targeted by this array allowing the screening of a large number of strains in a limited time and at an affordable cost. The relatively short turnaround time (~8 h) and the use of a software source code freely available allow rapid data acquisition and analysis. In addition, the method is cost-effective with a reagent price of 18 €/sample.
The designed array targeted AMR from 9 antimicrobial families, including resistance to critically important antimicrobials, linezolid, and vancomycin, which are important to monitor in both human and animal sectors. Due to the flexibility of the method, a more specific bead array (e.g., linezolid-array) could also be easily designed by removing/adding probes to respond to a particular epidemiologic situation (i.e., location) or to screen other bacterial species; and could also be extended to other research areas (i.e., virulence). Animal isolates were investigated; nevertheless, other reservoirs of AMR genes (humans, environment) could be assessed with the array. Besides AMR genes, resistance resulting from point mutations in receptors (e.g., ciprofloxacin resistance) or in specific proteins (e.g., daptomycin resistance) are targetable as well. In parallel to the official monitoring based on AST, screening with the array allowed us to rapidly determine genetic profiles circulating among livestock animals in Belgium. Isolates of this study have frequently been shown to carry two or more resistance genes conferring the same resistance phenotype, and sometimes genes from the same family, for example erm or tet genes. This accumulation of genes has been frequently observed while a single gene is sufficient to confer resistance, and may be explained by acquisition at different times and/or under different conditions, including their possible colocation on plasmids carrying multiple resistance genes even in absence of selective pressure . Many studies suggested that commensal bacteria are probable reservoirs for AMR genes and can transfer these to pathogenic organisms, for example Bacillus spp. or Salmonella spp. (Frye & Jackson, 2013;Schwarz et al., 2018). Also, while the spread of resistance genes such as ermB and tetM is limited to Gram-positive bacteria, genes such as aadE or tetL are found both in Gram-positive such as reported here but also in Gram-negative bacteria (E. coli) as reported elsewhere (Frye & Jackson, 2013;Schwarz et al., 2018). This array allowed the identification of AMR genes in resistant isolates, spotting a few isolates without genetic explanation as interesting candidates for WGS to identify new resistance mechanisms. This approach allowed us to demonstrate the presence of vanL in one E. faecalis isolate from animal origin. Our study also highlighted the presence of the lnuB gene in staphylococci, rarely reported in the literature. Finally, the missing concordance between aminoglycoside resistance genes and phenotypic resistance observed in this study illustrates the importance of still relying on the routine phenotypic susceptibility test for resistance monitoring. Oppositely, the presence of an oxazolidinone resistance gene in a susceptible isolate occurred several times and showed the importance of genotyping as a complement to phenotyping, particularly for such potentially inducible genes. Our data stress the importance of interpreting AMR with caution and the complementarity of both phenotyping and genotyping methods.

ACKNOWLEDGMENTS
We thank the Federal Agency for the Safety of the Food Chain for collecting the samples and isolates used in this study. We thank A.
Radu for technical support, technicians of the service Transversal Activities in Applied Genomics at Sciensano, Belgium for performing Next Generation Sequencing runs and the development and maintenance of the in-house instance of the Galaxy workflow management system. We also thank V. Perreten, A. J. O'Neill, S. Schwarz, F. Fux of the Bacterial Diseases Unit at Sciensano, and the EURL-AR for providing strains used for the array validation.

CONFLICT OF INTEREST
None declared.

DATA AVAILABILITY STATEMENT
All data analyzed during this study are included in this published article. Strains with source as "In-house collection" were isolates sequenced by whole-genome sequencing of our collection.